Down regulation of estrogen receptor (ERalpha) is a fundamental regulatory mechanism by which estrogen controls cellular response to stimulation. In breast cancer cells, the levels of ERalpha can often be inappropriately elevated or undetectable, which can affect both the natural history and treatment of the disease. Our laboratory and others have discovered that the most acute mechanism by which estrogen controls cellular ERalpha content is the regulated destruction of receptor protein by the 26S proteasome. Recent evidence suggests that proteasome activity is crucial for activation of transcription by steroid hormone receptors. The specific role that ERalpha proteolysis plays in the control of estrogen-dependent transcription, however, is unknown. We hypothesize that the regulation of ERalpha proteolysis and transactivation are functionally coupled events. Whereas proteolysis can control the magnitude of receptor transcription by affecting the kinetics of transcriptional complex formation on DNA, events within transcriptional complex assembly can reciprocally mark the pool of receptors destined for destruction. To address this hypothesis, Aim 1 seeks to identify a sequence element in the ERalpha N-terminus required for estrogen-induced proteolysis by mutagenesis and peptide inhibitor studies; Aim 2 proposes to define the specific role of ERa proteolysis and the general requirement for proteasome function in ERalpha transcriptional complex assembly using chromatin immunoprecipitation analyses; and, Aim 3 seeks to elucidate mechanism(s) that protect ERalpha from proteolysis by dissecting the activity of thyroid hormone, an endogenous inhibitor of ERalpha degradation and transcription. Through the successful completion of the herein proposed experiments, we hope to delineate the regulatory events that link the control of ERalpha proteolysis and transcription. Understanding the relationship between receptor proteolysis and transcription will likely contribute to the identification of novel targets for therapeutic intervention of ERalpha-dependent malignancies.